Compounds for use in the treatment of cancer

ABSTRACT

Pharmaceutical compositions comprise a first compound of Formula I or a physiologically acceptable salt thereof, and a cyto-protective amount of a manganese chelate of N,N′-dipyridoxyl ethylenediamine-N,N′-diacetic acid (MnPLED): 
                         
wherein R 1 , R 2 , R 3 , R 4  and X are defined in the specification.

FIELD OF INVENTION

The present invention relates to a compound for use in the treatment ofcancer. The invention also relates to the use of the compound in themanufacture of a medicament for the treatment of cancer. Also disclosedis a method of treatment of cancer in the human or non-human bodywherein said method comprises administrating to said body a compound asmentioned above. The invention further relates to a pharmaceuticalcomposition comprising the above mentioned compound and a compoundhaving cyto-protective ability. The invention also relates to the use ofthe pharmaceutical composition in the manufacture of a medicament fortreatment of cancer.

BACKGROUND

EP0910360, U.S. Pat. No. 6,147,094, EP0936915, U.S. Pat. No. 6,258,828,EP1054670, U.S. Pat. No. 6,310,051, EP1060174, U.S. Pat. No. 6,391,895disclose the use of dipyridoxyl based chelating agents and their metalchelates and the use of certain manganese containing compounds, inparticular manganese chelates, in medicine. The use of such compounds ascell protective agents in cancer therapy is also disclosed. The abovecited documents disclose that certain chelating agents, in particulardipyridoxyl and aminopolycarboxylic acid based chelating agents, andtheir metal chelates are effective in treating or preventinganthracycline-induced cardiotoxicity, ischaemia-reperfusion-inducedinjuries and atherosclerosis. Dipyridoxyl based chelating agents andtheir chelates with trivalent metals have previously been described byTaiferro (Inorg. Chem. 1984; 23:1183-1192).

DPDP (N,N′-bis-(pyridoxal-5-phosphate)-ethylenediamine-N,N′-diaceticacid), and the dephosphorylated counterpart PLED (N,N′-dipyridoxylethylenediamine-N,N′-diacetic acid) are dipyridoxyl compounds able tochelate metals. It has previously been described that the manganesechelates of these compounds, MnDPDP and its dephosphorylated counterpartMnPLED, possess catalytic antioxidant activity, i.e., a superoxidedismutase (SOD) mimetic activity. These compounds have been shown tohave a protective effect in normal cells e.g., against the cytostaticdrug doxorubicin and ischemia-reperfusion. It is the SOD mimeticactivity, which is an inherent property of redox-active manganese(Mn²⁺/Mn³⁺) bound to DPDP/PLED (Brurok et al., Biochem Biophys ResCommun. 1999; 254:768-721), that explains the protective effects.Consequently, Brurok and co-workers (1999) have shown that the PLEDmetal complex loses its catalytic activity after replacing redox-activemanganese with redox-inactive zinc (Zn²⁺).

Laurent et al. (Cancer Res. 2005; 6:948-56) and Alexandre et al., (JNatl Cancer Inst. 2006; 98:236-44) have recently described that MnDPDP(equivalent to the ready-to-use MRI contrast agent Teslascan) not onlyincreased survival of normal cells but also increased cancer cell deathduring cytostatic treatment, e.g., with oxaliplatin. Cytostatic drugsmay cause cancer cell death by elevating intracellular H₂O₂ and inducingapoptosis. The Laurent et al., hypothesis was that MnDPDP due to its SODmimetic activity elevated intracellular H₂O₂ and hence acted in synergywith cytostatic drugs. Since the basal level of H₂O₂ is much lower innormal cells compared to cancer cells, the authors suggested thatelevation from a low H₂O₂ level induced cell survival in normal cells.They furthermore suggested that elevation from a much higher basal levelof H₂O₂ in cancer cells at the same time resulted in apoptoticsignalling and hence cell death. Consequently, these authors suggestedthat both these effects, i.e., the increase in cancer cell death andsurvival of normal cells, were caused by the SOD mimetic activity ofMnDPDP, an activity which is absolutely dependent on redox-activemanganese. It has also been shown that intravenous injection of both themother compound MnDPDP and its metabolite MnPLED into mice gave rise toprotection against certain cytostatic drugs (EP0910360 and U.S. Pat. No.6,147,094).

When MnDPDP is intravenously injected into humans about 70% of theadministered manganese is released. For diagnostic imaging use and foroccasional therapeutic use, dissociation of manganese from MnDPDPrepresents no major problem. However, for more frequent use accumulatedmanganese toxicity may represent a serious toxicological problem,particularly when it comes to neurotoxicity (Crossgrove & Zheng; NMRBiomed. 2004; 17:544-53). Thus, for frequent therapeutic use, as incancer treatment, compounds that dissociate manganese should be avoided.

A number of anti-tumour agents are associated with adverse side effects.Paclitaxel, for example, is one such cytostatic drug which has shownanti-neoplastic activity against a variety of malignant tissues,including those of the breast. However, at the dosages required to havean anti-neoplastic effect, paclitaxel has a number of adverseside-effects which include cardiovascular irregularities as well ashaematological and gastrointestinal toxicity. Oxaliplatin, in particularin combination with 5-fluorouracil (5-FU), is another example of acytostatic drug that is effective in the treatment of colorectal cancerbut its use is restricted by severe adverse side-effects, in particularhaematological toxicity and neurotoxicitiy. Severe side-effects alsorestrict the use of radiation therapy in cancer.

There is hence an unmet medical need to find new chemotherapeutic drugswith fewer side-effects, in addition to find methods to protect normalcells against injuries caused by cancer treatment.

DESCRIPTION OF THE INVENTION

The present invention provides a compound with cancer cell-killingability for use in the treatment of cancer. The invention also providesthe use of a compound of the invention in the manufacture of amedicament for treatment of cancer. The invention further comprises amethod of treatment of cancer in the human or non-human body, whereinsaid method comprises administrating to said body a compound of theinvention. Also disclosed is a pharmaceutical composition whichcomprises the above mentioned compound and a second compound having acyto-protective ability, i.e. the ability to protect normal cells duringcancer treatment from the side-effects caused by chemotherapeutic drugsand radiation. Also provided is the use of a pharmaceutical compositionaccording to the invention in the manufacture of a medicament fortreatment of cancer. The invention also relates to a method of treatmentof cancer in the human or non-human body, wherein said method comprisesadministrating to said body the compound of the invention.

A first aspect of the invention is directed to a compound of Formula I

or a salt thereof, for use in the treatment of cancer, wherein

-   X represents CH or N,-   each R¹ independently represents hydrogen or —CH₂COR⁵;-   R⁵ represents hydroxy, optionally hydroxylated alkoxy, amino or    alkylamido;-   each R² independently represents a group ZYR⁶; Z represents a bond,    or a C₁₋₃ alkylene or oxoalkylene group optionally substituted by a    group R⁷;-   Y represents a bond, an oxygen atom or a group NR⁶;-   R⁶ is a hydrogen atom, a group COOR⁸, an alkyl, alkenyl, cycloalkyl,    aryl or aralkyl group optionally substituted by one or more groups    selected from COOR⁸, CONR⁸ ₂, NR⁸ ₂, OR⁸, ═NR⁸, ═O, OP(O) (OR⁸)R⁷    and OSO₃M;-   R⁷ is hydroxy, an optionally hydroxylated, optionally alkoxylated    alkyl or aminoalkyl group;-   R⁸ is a hydrogen atom or an optionally hydroxylated, optionally    alkoxylated alkyl group;-   M is a hydrogen atom or one equivalent of a physiologically    tolerable cation; e.g. an alkali or alkaline earth cation, an    ammonium ion or an organic amine cation, such as meglumine ion;-   R³ represents a C₁₋₈ alkylene group, preferably a C₁₋₆, e.g. a C₂₋₄,    alkylene group, a 1,2-cykloalkylene group, or a 1,2-arylene group,    optionally substituted with R⁷; and each R⁴ independently represents    hydrogen or C₁₋₃ alkyl and wherein the compound is optionally a    chelate with one or two Na⁺ or K⁺, but a combination of one Na⁺ and    one K⁺ is also possible.

In an embodiment of the invention R⁵ is hydroxy, C₁₋₈ alkoxy, ethyleneglycol, glycerol, amino or C₁₋₈ alkylamido;

-   Z is a bond or a group selected from CH₂, (CH₂)₂, CO, CH₂CO,    CH₂CH₂CO or CH₂COCH₂; Y is a bond;-   R⁶ is a mono- or poly(hydroxy or alkoxylated) alkyl group or a group    of the formula OP(O)(OR⁵)R⁷; and R⁷ is hydroxy, or an unsubstituted    alkyl or aminoalkyl group.

In another embodiment of the invention R³ is ethylene and each group R¹represents —CH₂COR⁵ in which R⁵ is hydroxy.

In yet another embodiment of the invention the compound of Formula I isN,N′-dipyridoxyl ethylenediamine-N,N′-diacetic acid (PLED).

In a further embodiment of the invention the compound of Formula I isN,N′-bis-(pyridoxal-5-phosphate)-ethylenediamine-N,N′-diacetic acid(DPDP).

In another embodiment of the invention is described the use of acompound of Formula I, as defined above, in the manufacture of amedicament for treatment of cancer. The cancer may be any type of cancere.g. leukaemia, breast cancer, colorectal cancer, liver cancer andmetastases thereof. The medicament may comprise pharmaceuticallyacceptable carriers or excipients.

A second aspect of the invention is directed to a method of treatment ofcancer in the human or non-human body, said method comprisingadministrating to said body a compound of Formula I according to theinvention.

A third aspect of the invention is directed to a pharmaceuticalcomposition comprising a first compound of Formula I, as definedhereinabove, and a second compound having a cyto-protective ability.

In another embodiment of the invention the second compound comprised inthe pharmaceutical composition is a metal chelate comprising a compoundof Formula I as defined above.

In yet another embodiment of the invention the metal chelate comprisedin the pharmaceutical composition has a K_(a) value preferably in therange of from 10⁸ to 10²⁴, more preferably in a range of from 10¹⁰ to10²² and most preferably in the range of from 10¹² to 10²⁰.

In a yet further embodiment of the invention the metal chelate comprisedin the pharmaceutical composition has a lower K_(a) value than the K_(a)value of an iron (Fe³⁺) chelate comprising a compound of Formula I asdefined above, by a factor of at least 10³.

In still another embodiment of the invention the metal in the metalchelate comprised in the pharmaceutical composition is manganese (Mn²⁺or Mn³⁺) or copper (Cu⁺ or Cu²⁺).

In another embodiment of the invention the first compound of thepharmaceutical composition is N,N′-dipyridoxylethylenediamine-N,N′-diacetic acid and the second compound is a metalchelate comprising N,N′-dipyridoxyl ethylenediamine-N,N′-diacetic acid.The metal in the metal chelate is preferably manganese or copper.

In a preferred embodiment of the invention the first compound of thepharmaceutical composition isN,N′-bis(pyridoxal-5-phosphate)-ethylenediamine-N,N′-diacetic acid andthe second compound is a metal chelate comprising N,N′-dipyridoxylethylenediamine-N,N′-diacetic acid. The metal in the metal chelate ispreferably manganese or copper.

In a further embodiment of the invention the second compound of thepharmaceutical composition according to the invention may preferablyconstitute 1/100 to 99/100 of the first compound, at a molar basis.

In a yet further embodiment of the invention is provided thepharmaceutical composition according to the invention for use in thetreatment of cancer.

In a fourth aspect of the invention is provided a kit comprising apreparation of a first active ingredient, which is a compound of FormulaI as defined above, a preparation of a second active ingredient, whichis a metal chelate comprising a compound of Formula I as defined above,and optionally instructions for the simultaneous, sequential or separateadministration of the preparations to a patient in need thereof.

In a fifth aspect of the invention is provided the use of apharmaceutical composition according to the invention in the manufactureof a medicament for treatment of cancer. The cancer may be any type ofcancer e.g. leukaemia, breast cancer, colon cancer, liver cancer andmetastases thereof.

In another embodiment of the invention is provided the use of apharmaceutical composition according to the invention, wherein themedicament further comprises pharmaceutically acceptable carriers orexcipients.

In a sixth aspect of the invention is provided a method of treatment ofcancer in a patient in need of such treatment, comprising the step ofadministrating to said patient a cancer inhibiting amount of apharmaceutical composition according to the invention, optionally incombination with pharmaceutically acceptable carriers and excipients.

In a further embodiment of the invention is provided a method whereinthe pharmaceutical composition is administered together with one or moreother anti-cancer drug(s). The anti-cancer drug could be any anticancerdrug, e.g., doxorubicin, epirubicin, oxaliplatin, cisplatin,carboplatin, paclitaxel, 5-fluorouracil, cyclophosphamide, gemcitabine,irinotecan, and methotrexate.

In another embodiment of the invention is provided a method, wherein thepharmaceutical composition as described above and the one or more otheranti-cancer drug(s) are administered simultaneously, separately orsequentially to said patient.

In a further embodiment of the invention is provided a method oftreatment as described above, wherein the treatment is combined withradiation therapy.

The invention should also be understood to include the use of thepharmaceutical composition according to the invention in the manufactureof a medicament for treatment of cancer

The compounds of Formula I as defined above for use in the inventionshould be understood to be therapeutically active and physiologicallypreferred compounds.

As used herein the terms “alkyl” and “alkylene” include straight-chainedand branched, saturated and unsaturated hydrocarbons. The term“1,2-cykloalkylene” includes both cis and trans cycloalkylene groups andalkyl substituted cycloalkylene groups having from 5-8 carbon atoms. Theterm “1,2-arylene” includes phenyl and naphthyl groups and alkylsubstituted derivatives thereof having from 6 to 10 carbon atoms.

Unless otherwise specified, any alkyl, alkylene or alkenyl moiety mayconveniently contain from 1 to 20, preferably 1-8, more preferably 1-6and especially preferably 1-4 carbon atoms.

Cycloalkyl, aryl and aralkyl moieties may conveniently contain 3-18,preferably 5-12 and especially preferably 5-8 ring atoms. Aryl moietiescomprising phenyl or naphthyl groups are preferred. As aralkyl groups,phenyl C₁₋₈ alkyl, especially benzyl, are preferred.

Where groups may optionally be substituted by hydroxyl groups, this maybe monosubstitution or polysubstituition and, in the case ofpolysubstitution, alkoxy and/or hydroxyl substituents may be carried byalkoxy substitutents.

In Formula I, R⁵ is preferably hydroxyl, C₁₋₈ alkoxy, ethylene glycol,glycerol, amino or C₁₋₈ alkylamido. Preferably each group R¹ represents—CH₂COR⁵ in which R⁵ is hydroxy.

In the compound of Formula I, Z is preferably a bond or a group selectedfrom CH₂, (CH₂)₂, CO, CH₂CO, CH₂CH₂CO or CH₂COCH₂. Preferably, Yrepresents a bond.

The compound of Formula I may have the same or different R² groups onthe two pyridyl rings and these may be attached at the same or differentring positions. However, it is especially preferred that substitution beat the 5- and 6-positions, most especially the 6-position, i.e. para tothe hydroxyl group. Compound in which the R² groups are identical andidentically located, e.g. 6,6′, are especially preferred.

Preferred as groups R⁶ are mono- or poly(hydroxy or alkoxylated) alkylgroups or a group of the formula OP(O)(OR⁶)R⁷.

R⁷ is preferably hydroxyl or an unsubstituted alkyl or aminoalkyl group.

Particularly preferred identities for group R² includeCHR⁷OCO(CH₂)_(X)Ph and CHR⁷OCO(CH₂CO)_(x)Ph (wherein x is 1 to 3),CHR⁷OCOBu^(t), CH₂N(H)R^(6′), CH₂N(H)R^(6′), N(H)R^(6′), N(R^(6′))₂,CH₂OH, CH₂OR^(6′), COOR^(6′), CON(H)R^(6′), CON(R^(6′))₂ or OR^(6′)(where R^(6′) is a mono- or polyhydroxylated, preferably C₁₋₄,especially preferably C₁₋₃, alkyl group), (CH₂)_(n)COOR^(7′) (wherein nis 1 to 6), COOR^(7′) (where R⁷′ is a C₁₋₄ alkyl, preferably C₁₋₃,especially preferably a methyl group), CH₂OSO₃ ⁻M, CH₂CH₂COOH,CH₂OP(O)(OH)(CH₂)₃NH₂, CH₂OP(O)(OH)CH₃ or CH₂OP(O)(OH)₂ group. Yet morepreferably, R² represents a group of the formula CH₂OP(O)(OH)₂.

Compounds of Formula I in which R³ is ethylene and R² has any of theidentities listed above are particularly preferred.

The pharmaceutical composition of the present invention and thepreparations included in the kit of the present invention may beformulated with conventional pharmaceutical or veterinary formulationaids, for example stabilizers, antioxidants, osmolality adjustingagents, buffers, pH adjusting agents etc. and may be in a form suitablefor parenteral or enteral, administration, for example injection orinfusion. Thus the pharmaceutical composition of the present inventionmay be in a conventional pharmaceutical administration form such as atablet, capsule, powder, solution, suspension, dispersion, syrup,suppository, etc.

The compounds of Formula I and the metal chelates comprising thecompounds of Formula I may therefore be formulated for administrationusing physiologically acceptable carriers and/or excipients in a mannerwell-known to those skilled in the art. The compounds of Formula I andthe metal chelates comprising the compounds of Formula I may for examplebe suspended or dissolved in an aqueous medium, optionally with theaddition of pharmaceutically acceptable excipients.

The medicament and the pharmaceutical composition according to thepresent invention may be administered by various routes, for exampleorally, transdermally, rectally, intrathecally, topically or by means ofinhalation or injection, in particular subcutaneous, intramuscular,intraperitoneal or intravascular injection. Other routes ofadministration may be envisioned if they increase the effectiveness, thebioavailability or the tolerance of the products. The most appropriateroute can be chosen by those skilled in the art according to theformulation used.

The cancer inhibiting amount of a medicament administered to a patientis dependent on several different factors such as the type of cancer,the age and weight of the patient, etc., and the attending physicianwill follow the treatment to adjust the doses if necessary based onlaboratory tests.

Generally doses of the active compounds, i.e., first and secondcompound, in the pharmaceutical composition according to the inventionwill comprise between 0.01 μmol of the compounds per kilogram of thepatient's body weight to 100 μmol of the compound per kilogram of thepatient's body weight.

The pharmaceutical composition of the invention may thus comprise acompound of Formula I, in particular DPDP or its dephosphorylatedcounterparts DPMP and PLED, representing a method for treating variouscancer diseases, alone or in combination with other cytostatic drugs orradiotherapy.

If not all of the labile hydrogens of the chelates according to theinvention are substituted by the complexed metal ion, biotolerabilityand/or solubility of the chelates may be increased by substituting theremaining labile hydrogen atoms with physiologically biocompatiblecations of inorganic and/or organic bases or amino acids. Examples ofsuitable inorganic cations include Li⁺, K⁺, Na⁺ and especially Ca²⁺.Suitable organic cations include ammonium, substituted ammonium,ethanolamine, diethanolamine, morpholine, glucamine, N,N,-dimetylglucamine, lysine, arginine or ornithine.

Where the first or the second compound according to the inventioncarries an overall charge it may conveniently be used in the form of asalt with a physiologically acceptable counterion, for example anammonium, substituted ammonium, alkali metal or alkaline earth metal (e.g. calcium) cation or an anion deriving from an inorganic or organicacid. In this regard, meglumine salts are particularly preferred.

The therapeutic agents of the present invention may be formulated withconventional pharmaceutical or veterinary formulation aids, for examplestabilizers, antioxidants, osmolality adjusting agents, sweeteningagents etc.

As previously described the invention provides a compound of Formula Ias defined above for use in the treatment of cancer. When the presentinventors compared MnDPDP and DPDP they surprisingly found that DPDP wasmore efficacious than MnDPDP in its ability to kill cancer cells andthey concluded that the previously described cancer cell killing abilityof MnDPDP is an inherent property of DPDP. The invention thus provides anew compound for use in treatment of cancer while avoiding the problemof toxicity related to manganese release.

The compound may, as previously mentioned, also be used in combinationwith a second compound having cyto-protective ability. In an embodimentof the invention is described the use of a metal chelate comprising thecompound of Formula I as the compound having the cyto-protectiveability. This metal chelate is surprisingly found to be much more stablethan MnDPDP and the problem of metal release is thereby avoided. Asuitable drug combination for cancer-treatment is thus presented.

The stability of MnDPDP after administration into man is according toprior art mainly governed by the stability constants between DPDP andMn²⁺ and other competing metals, mainly non-redox active Zn²⁺ which hashigher affinity for DPDP than Mn²⁺ (Rocklage et al., Inorg Chem 1989;28:477-485 and Toft et al., Acta Radiol 1997; 38:677-689). Afterintravenous injection in man, in addition to dissociation of Mn²⁺, thetwo phosphates are hydrolyzed from DPDP, giving rise to PLED. Shortlyafter intravenous injection about 30% of the injected MnDPDP istransformed into MnPLED, and according to prior art (Toft et al., 1997)Mn²⁺ will also dissociate from PLED, actually more readily than fromDPDP. Such behaviour of MnPLED is highly supported by the reportedstability constants in the literature (Rocklage et al., 1989).

However, reinterpretation of previously published results may in factsuggest that MnPLED is much more stable than MnDPDP (regarding metalstability) during in vivo conditions. If human plasma concentration datataken from the study by Toft et al. 1997 is recalculated it is seen thatdisappearance of MnDPDP and its 5 metabolites from the plasma roughlyparallels that of MnPLED between 30 and 60 minutes (after the initialdistribution phase). All these compounds are eliminated from the bodythrough renal excretion, and if manganese dissociated from MnPLED onewould expect that these two processes diverged during that period oftime. This finding may suggest that MnPLED is stable during in vivoconditions.

Taking the reported stability constants for Mn²⁺ and Fe³⁺ inconsideration, the results in Example 3 quite clearly further supportsthe paradoxical suggestion that MnPLED is much more stable than MnDPDPwhen it comes to dissociation of Mn²⁺. It may furthermore be anticipatedfrom the pharmacokinetic data that target cells and tissues will not beexposed for concentrations higher than 5 μM of MnPLED, i.e.,concentrations where MnPLED are expected to be stable.

The present invention shows that MnPLED is much more stable than MnDPDP,and most importantly, by using MnPLED instead of its mother substanceMnDPDP, it may be possible to circumvent the serious toxicologicalmanganese problem evident at frequent therapeutic use in man.

It should furthermore be stressed that pretreatment with MnPLED in micehas shown to be approximately 100 times more efficacious than MnDPDP(EP0910360 and U.S. Pat. No. 6,147,094). This suggests that the MnPLEDdose could be considerable lowered in comparison to MnDPDP, which wouldfurther reduce the toxicological potential of the pharmaceuticalcomposition, and hence increase the therapeutic index further. Moreover,a lower dose of MnPLED (3 μmol/kg) than that employed in MnDPDP-enhanceddiagnostic imaging (5-10 μmol/kg) has been shown to reduce infarct sizein pigs (Karlsson et al., Acta Radio) 2001; 42:540-547), and even muchlower doses have been demonstrated to be effective in the same animalmodel (unpublished data).

Interestingly, MnDPDP did not reduce the infarct size in pigs. This ispresumably due to a much faster replacement of manganese for zinc inpigs compared to man. Ten minutes after injection of MnDPDP allmanganese has been replaced with zinc (Karlsson et al., 2001), whichdiffers from man (and some other investigated species) where about 30%of the injected manganese stays bound to the chelator for a considerableamount of time. As mentioned previously, the protection of normal cells,in this case myocardial cells, is dependent on redox-active manganese.According to prior art (Rocklage et al., 1989), the stability constantbetween Mn²⁺ and DPDP is 15.10 (log K), whereas the stability constantbetween Zn²⁺ and DPDP is 18.95, i.e., Mn²⁺ dissociates about 1000 timesmore readily than Zn²⁺ from DPDP. The corresponding stability constantsbetween Mn²⁺ and PLED and Zn²⁺ and PLED are 12.56 and 16.68,respectively, i.e., Mn²⁺ once again dissociates about 1000 times morereadily than Zn²⁺. From this and the published metabolic scheme (Toft etal., 1997) one would not expect any major difference in stabilitybetween MnDPDP and MnPLED, in respect to exchange of manganese for zinc,after administration into pigs. The above mentioned infarct reductionseen after administration of MnPLED, but not after MnDPDP, is hence aparadoxical finding. However, the present invention as exemplified inPatent Example 3 comes up with a reasonable explanation to the paradox,namely that MnPLED is a more stable complex than MnDPDP, and mostimportantly it solves the toxicological problems of manganeseinstability.

An advantage of combining the DPDP's anticancer activity with MnPLED'scyto-protective activity with regard to normal cells and tissue may beexemplified by the problem of using dexrazoxane as a cardioproteciveagent against anthracycline-induced cardiotoxicity. Although far fromevident, dexrazoxane is not recommended at the beginning of theanthracycline therapy in patients with metastatic breast cancer becauseof the possibility of reducing the anticancer effect of theanthracyclines (Yeh et al., Circulation 2004; 109:3122-3132). However,as has been demonstrated for MnDPDP by the present inventors and others,preclinical data quite clearly shows that this is not a problem when itcomes to our approach. One conceivable explanation to this is the twodistinct and inherent activities of MnDPDP, namely its anticanceractivity and its cytoprotective activity, and which in our invention hasbeen further separated into two distinct chemical entities, namely DPDP,possessing the anticancer activity, and MnPLED, possessing thecytoprotective activity in normal cells and tissues.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. MTT assay on human colon cancer SW480 cells in absence (control)and presence of MnDPDP, DPDP, oxaliplatin or DPDP+oxaliplatin (mean±SD;n=3).

FIG. 2. MTT assay on murine lymphoma J774 cells in absence (control) andpresence of MnDPDP, DPDP, oxaliplatin or DPDP+oxaliplatin (mean±SD;n=3).

FIG. 3. The cytostatic effect of increasing concentration of oxaliplatinin SW620, HCT-8 and hTERT-RPE1 cells (A). The cytostatic effect of a lowconcentration oxaliplatin alone or in combination with MnDPDP or DPDP inSW620 (B), HCT-8 (C) hTERT-RPE1 (D) cells (mean±SD; n=3).

FIG. 4. The Fenton assay in the presence of various concentrations DPDP,MnDPDP and MnPLED (mean±SD; n=3).

(A) Controls were run in parallel and at the end of the experiments(mean±SD; n=3).

(B) The controls also included absence of iron (—Fe), presence of theiron chelator desferrioxamine (15 μM DFO) or the hydroxyl radicalscavenger DMS0 (10% DMSO) (mean±SD; n=3).

EXAMPLES

The invention will now be further demonstrated and described by thefollowing non-limiting examples. The examples should be understood toonly exemplify the invention and the invention should not be limitedthereto.

Example 1

The cytostatic activity of DPDP and MnDPDP was compared by co-incubatinghuman colon cancer cells (SW480) and murine lymphoma cells (J774) withMnDPDP, DPDP and/or oxaliplatin.

Method

The viability of cells was measured using the MTT assay. Briefly, 20,000SW480 or J774 cells were seeded per well on a 96-well plate and grownover night in Dulbecco's Modified Eagle's Medium (DMEM) containing 10%fetal bovine serum, 2 mM L-glutamine, 100 UI/ml penicillin and 100 μg/mlstreptomycin at 37° C. in humidified air with 5% CO₂. Cells were thenexposed for 24 h to 50 μM MnDPDP, 50 μM DPDP or 10 μM oxaliplatin at 37°C. The effect of combining 50 μM DPDP with 10 μM oxaliplatin onviability was also tested. The viability of the cells was then assessedby adding 5 mg/ml methylthiazoletetrazolium (MTT) to a finalconcentration of 0.5 mg/ml and incubating cells for a further 4 h at 37°C. The blue formazan that is formed by mitochondrial dehydrogenases ofviable cells was then dissolved over night at 37° C. by adding 10% SDSand 10 mM HCl to a final concentration of 5% SDS and 5 mM HCl. Finally,the absorbance of the solution was read at 570 nm with a reference at670 nm in a microplate reader Spectramax 340 (Molecular Devices,Sunnyvale, Calif., USA) connected to an Apple Macintosh computer runningthe program Softmax Pro V1.2.0 (Molecular Devices, Sunnyvale, Calif.,USA).

Results

The cytostatic activity of 50 μM DPDP was statistically significant moreefficacious than that of 50 μM MnDPDP in human colon SW480 cells(unpaired t-test) (FIG. 1). There was a tendency, although notstatistically significant (p<0.07), that DPDP in combination withoxaliplatin was more potent than oxaliplatin alone in SW480 cells. Thecorresponding cytostatic activity of 50 μM MnDPDP and 50 μM DPDP inmurine lymphoma J774 cells is presented in FIG. 2, and as obvious fromthis figure, DPDP was significantly more efficacious to kill thelymphoma cells than MnDPDP. Furthermore, DPDP in combination withoxaliplatin was significantly more efficacious than oxaliplatin alone.

Conclusion

When MnDPDP and DPDP were compared it was surprisingly found that DPDPwas more efficacious than MnDPDP in its ability to kill cancer cells,and it is concluded that the previously described cancer cell killingability of MnDPDP is an inherent property of the DPDP.

Example 2

The cytostatic activity of MnDPDP or DPDP in the absence and presence ofoxaliplatin was tested in human adenocarcinoma cells (SW620), humanileocecal colorectal adenocarcinoma cells and normal retinal epitheliatelomerase immortalized cells (hTERT-RPE1).

Method

HCT-8 cells were grown in RPMI 1640 medium with sodium pyruvate (1 mM)and 10% horse serum. SW620 were grown in ATCC-formulated Leibovitz'sL-15 Medium with 10% fetal bovine serum. Cells were kept at 37° C. in ahumidified atmosphere containing 5% carbon dioxide. The cells wereharvested in the log-phase for experimental use. The fluorometricmicroculture cytotoxicity assay (FMCA) was used to investigate thecytostatic activity of oxaliplatin, MnDPDP or DPDP and combinationsthereof. FMCA is based on measurement of fluorescence generated fromhydrolysis of fluorescein diacetate (FDA) to fluorescein by cells withintact plasma membranes. Briefly, 96-well microtiter plates were freshlyprepared with drug solutions in triplicates at 10 times the desired drugconcentrations. Cell suspensions were seeded into the drug-preparedplates with 20 000 cells per well and plates were then incubated for 72h. After incubation, the plates were washed, FDA was added, and after 50min of incubation the fluorescence generated (excitation 480 nm) wasmeasured at 538 nm in a fluorometer (Fluorostar Optima, BMGTechnologies). The fluorescence is proportional to the number of cellswith intact plasma membrane present in the well.

Results

The cytostatic activity of increasing concentrations of oxaliplatin incancer cells (SW620 and HCT-8) and normal cells (hTERT-RPE1) ispresented in FIG. 3A. Oxaliplatin demonstrated aconcentrations-dependent cytostatic effect in both cancer cell lines butonly a slight effect at its highest concentration in normal cells.Neither MnDPDP nor DPDP demonstrated any cytostatic effect in any ofthese cells (not shown). However, 100 μM DPDP but not MnDPDPsignificantly (unpaired t-test) potentiated the cytostatic effects of alow concentration (8 μM) of oxaliplatin in both cancer cell lines butnot in normal cells (FIG. 3B-D).

Conclusion

When MnDPDP and DPDP were compared it was surprisingly found that DPDPwas much more efficacious than MnDPDP in its ability to increase thecancer-killing ability of oxaliplatin, and it is concluded that thepreviously described cancer cell killing ability of MnDPDP is aninherent property of the DPDP.

Example 3

By permitting the chelator DPDP, the metal complexes MnDPDP and MnPLEDto compete with iron in the Fenton assay, the stability of MnDPDP andMnPLED was compared.

Method

Ferric iron (10 μM) was partially reduced to its ferrous form bycysteine (100 μM) in 150 mM acetate buffer. H₂O₂ (100 μM) was added toinitiate the production of hydroxyl radicals (HO.). The latter oxidizeH₂DCF (non-fluorescent 2′,7′-dichlorodihydrofluorescein; 5 μM) tofluorescent DCF (2′,7′-dichlorofluorescein). H₂DCF was obtained byhydrolysing its acetate ester (H₂DCF-DA)]. DMSO (10%) and DFO (10 μM)were used to demonstrate the formation of HO. and the involvement ofiron respectively. DPDP, MnDPDP and MnPLED at various concentrationswere assayed for their iron-chelating capacity. Fluorescence wasmeasured in an FL600 Microplate Fluorescence reader (Bio-Tek, Winooski,Vt., U.S.A.) at ex 485 nm and em 530 nm.

Results

FIG. 4 demonstrates that DPDP inhibited the Fenton reaction in adose-dependent manner; the inhibition started at 0.1 μM and wascompleted at 10 μM. The inhibitory pattern is in accordance with thereported high affinity of DPDP for ferric iron (log K=33.52). However,in the case of MnPLED no inhibition was evident up to and including aconcentration of 5 μM but at 10 μM the inhibition was complete. Theiron-chelating capacity of MnDPDP was significantly higher than that ofMnPLED.

Conclusion

The present results are in opposite to and highly surprising to what onewould expect from both the reported iron- and manganese-chelatingcapacity of MnDPDP and MnPLED. The reported stability constants betweenFe³⁺ and DPDP and between Fe³⁺ and PLED are 33.52 and 36.88 (log K),respectively, whereas the reported stability constants between Mn²⁺ andDPDP and Mn²⁺ and PLED are 15.10 and 12.56, respectively (Rocklage etal., 1989). One would hence expect MnPLED to be a much better inhibitorof the Fenton reaction than MnDPDP.

The invention claimed is:
 1. A pharmaceutical composition comprising afirst compound of Formula I or a physiologically acceptable saltthereof, and a cyto-protective amount of a manganese chelate ofN,N′-dipyridoxyl ethylenediamine-N,N′-diacetic acid (MnPLED):

wherein X is CH or N, each R¹ independently is hydrogen or —CH₂COR⁵; R⁵is hydroxy, optionally hydroxylated alkoxy, amino or alkylamido; each R²independently is a group ZYR⁶; Z is a bond, CO, or a C₁₋₃ alkylene oroxoalkylene group optionally substituted by a group R⁷; Y is a bond, anoxygen atom or a group NR⁶; R⁶ is a hydrogen atom, COOR⁸, an alkyl,alkenyl, cycloalkyl, aryl or aralkyl group optionally substituted by oneor more groups selected from COOR⁸, CONR⁸ ₂, NR⁸ ₂, OR⁸, ═NR⁸, ═O,OP(O)(OR⁸)R⁷ and OSO₃M; R⁷ is hydroxy, an optionally hydroxylated,optionally alkoxylated alkyl or aminoalkyl group; R⁸ is a hydrogen atomor an optionally hydroxylated, optionally alkoxylated alkyl group; M isa hydrogen atom or one equivalent of a physiologically tolerable cation;R³ is a C₁₋₈ alkylene group, a 1,2-cykloalkylene group, or a 1,2-arylenegroup, optionally substituted with R⁷; and each R⁴ independently ishydrogen or C₁₋₃ alkyl.
 2. The pharmaceutical composition of claim 1,wherein in the first compound of Formula I: R⁵ is hydroxy, C₁₋₈ alkoxy,ethylene glycol, glycerol, amino or C₁₋₈ alkylamido; Z is a bond or agroup selected from CH₂, (CH₂)₂, CO, CH₂CO, CH₂CH₂CO and CH₂COCH₂; Y isa bond; R⁶ is a mono- or poly(hydroxy or alkoxylated) alkyl group or agroup of the formula OP(O) (OR⁸)R⁷; and R⁷ is hydroxy, or anunsubstituted alkyl or aminoalkyl group.
 3. The pharmaceuticalcomposition of claim 1, wherein in the first compound of Formula I: R³is ethylene and each group R¹ represents —CH₂COR⁵ in which R⁵ ishydroxy.
 4. The pharmaceutical composition of claim 1, wherein the firstcompound is N,N′-dipyridoxyl ethylenediamine-N,N′-diacetic acid (PLED).5. The pharmaceutical composition of claim 1, wherein the first compoundis N,N′-bis-(pyridoxal-5-phosphate)-ethylenediamine-N,N′-diacetic acid(DPDP).
 6. The pharmaceutical composition of claim 5, wherein the molarratio of the MnPLED to DPDP is in the range of 1/100 to 99/100.
 7. Thepharmaceutical composition of claim 1, wherein the molar ratio of theMnPLED to the first compound is in the range of 1/100 to 99/100.
 8. Akit comprising a preparation of a first active ingredient of Formula I,a preparation of a manganese chelate of N,N′-dipyridoxylethylenediamine-N,N′-diacetic acid (MnPLED), and optionally instructionsfor the simultaneous, sequential or separate administration of thepreparations to a patient in need thereof:

or a physiologically acceptable salt thereof, wherein X is CH or N, eachR¹ independently is hydrogen or —CH₂COR⁵; R⁵ is hydroxy, optionallyhydroxylated alkoxy, amino or alkylamido; each R² independently is agroup ZYR⁶; Z is a bond, CO, or a C₁₋₃ alkylene or oxoalkylene groupoptionally substituted by a group R⁷; Y is a bond, an oxygen atom or agroup NR⁶; R⁶ is a hydrogen atom, COOR⁸, an alkyl, alkenyl, cycloalkyl,aryl or aralkyl group optionally substituted by one or more groupsselected from COOR⁸, CONR⁸ ₂, NR⁸ ₂, OR⁸, ═NR⁸, ═O, OP(O)(OR⁸)R⁷ andOSO₃M; R⁷ is hydroxy, an optionally hydroxylated, optionally alkoxylatedalkyl or aminoalkyl group; R⁸ is a hydrogen atom or an optionallyhydroxylated, optionally alkoxylated alkyl group; M is a hydrogen atomor one equivalent of a physiologically tolerable cation; R³ is a C₁₋₈alkylene group, a 1,2-cykloalkylene group, or a 1,2-arylene group,optionally substituted with R⁷; and each R⁴ independently is hydrogen orC₁₋₃ alkyl.
 9. The kit of claim 8, wherein in the first compound ofFormula I: R⁵ is hydroxy, C₁₋₈ alkoxy, ethylene glycol, glycerol, aminoor C₁₋₈ alkylamido; Z is a bond or a group selected from CH₂, (CH₂)₂,CO, CH₂CO, CH₂CH₂CO and CH₂COCH₂; Y is a bond; R⁶ is a mono- orpoly(hydroxy or alkoxylated) alkyl group or a group of the formula OP(O)(OR⁸)R⁷; and R⁷ is hydroxy, or an unsubstituted alkyl or aminoalkylgroup.
 10. The kit of claim 8, wherein in the first compound of FormulaI: R³ is ethylene and each group R¹ represents —CH₂COR⁵ in which R⁵ ishydroxy.
 11. The kit of claim 8, wherein the first compound isN,N′-dipyridoxyl ethylenediamine-N,N′-diacetic acid (PLED).
 12. The kitof claim 8, wherein the first compound isN,N′-bis-(pyridoxal-5-phosphate)-ethylenediamine-N,N′-diacetic acid(DPDP).
 13. The kit of claim 12, wherein the molar ratio of the MnPLEDto DPDP is in the range of 1/100 to 99/100.
 14. The kit of claim 8,wherein the molar ratio of the MnPLED to the first compound is in therange of 1/100 to 99/100.
 15. The pharmaceutical composition of claim 1,comprising a cancer-inhibiting amount of the first compound.
 16. The kitof claim 8, wherein the preparation of the first active ingredientcomprises a cancer-inhibiting amount of the first compound.